Protein renaturation microfluidic devices and methods of making and using the same

Abstract

Microfluidic devices having a protein renaturation component and methods for using the same are provided. Aspects of the present disclosure include microfluidic devices that include a separation medium with a first flow path and a protein renaturation component in fluid communication with the separation medium and having a second flow path. Also provided are methods of using the devices as well as systems and kits that include the devices. The devices, systems and methods find use in a variety of different applications, including diagnostic and validation assays.

That which is claimed is: 
     
       1. A microfluidic device comprising:
 a separation medium having a first flow path with a first flow direction; 
 a protein renaturation component in fluid communication with the separation medium and having a second flow path with a second flow direction; and 
 a binding medium in fluid communication with the separation medium and having a third flow path with a flow direction different from the second flow direction. 
 
     
     
       2. The microfluidic device according to  claim 1 , wherein the first flow path has a first directional axis and the second flow path has a second directional axis. 
     
     
       3. The microfluidic device according to  claim 1 , wherein the second flow path and the third flow path are in opposite directions along the second directional axis. 
     
     
       4. The microfluidic device according to  claim 1 , wherein the protein renaturation component comprises a sub-nanopore gel membrane. 
     
     
       5. The microfluidic device according to  claim 4 , wherein the sub-nanopore gel membrane is polymerized from a precursor having a monomer concentration ranging from 40 to 50% T. 
     
     
       6. The microfluidic device according to  claim 1 , wherein the binding medium comprises a binding member stably associated with a support. 
     
     
       7. The microfluidic device according to  claim 6 , wherein the binding member is a proteinaceous binding member. 
     
     
       8. The microfluidic device according to  claim 7 , wherein the proteinaceous binding member comprises an antibody or a binding fragment thereof. 
     
     
       9. The microfluidic device according to  claim 8 , wherein the proteinaceous binding member comprises a lectin. 
     
     
       10. The microfluidic device according to  claim 1 , wherein the device further comprises a loading medium in fluid communication with the separation medium. 
     
     
       11. The microfluidic device according to  claim 10 , wherein the device is configured so that the renaturation component bounds a first side of the separation medium, a binding medium bounds a second side of the separation medium that is opposite the first side, and the loading medium bounds a third side of the separation medium that is between the first and second sides. 
     
     
       12. The microfluidic device according to  claim 11 , further comprising a first set of side channels comprising the renaturation component, and a second set of side channels comprising the binding medium. 
     
     
       13. The microfluidic device according to  claim 11 , wherein the device is configured to apply first and second electric fields of differing directions to the separation medium. 
     
     
       14. The microfluidic device according to  claim 13 , wherein the first and second electric fields are orthogonal to each other. 
     
     
       15. The microfluidic device according to  claim 1 , further comprising a buffer. 
     
     
       16. The microfluidic device according to  claim 15 , wherein the buffer comprises a detergent. 
     
     
       17. The microfluidic device according to  claim 15 , further comprising a sample. 
     
     
       18. The microfluidic device according to  claim 17 , wherein the sample comprises an analyte of interest. 
     
     
       19. The microfluidic device according to  claim 18 , wherein the analyte comprises a fluorescent label. 
     
     
       20. The microfluidic device according to  claim 1 , wherein the separation medium is in direct contact with the protein renaturation component. 
     
     
       21. The microfluidic device according to  claim 1 , wherein the separation medium is in direct contact with the binding medium. 
     
     
       22. A kit comprising
 (a) a microfluidic device comprising:
 (i) a separation medium having a first flow path with a first flow direction; 
 (ii) a protein renaturation component in fluid communication with the separation medium and having a second flow path with a second flow direction; 
 (iii) a binding medium in fluid communication with the separation medium and having a third flow path with a flow direction different from the second flow direction; and 
 
 (b) a labeled binding member. 
 
     
     
       23. The kit according to  claim 22 , further comprising one or more reagents selected from the group consisting of a buffer, a detection reagent, a release reagent, a detergent, a refolding reagent and a denaturing reagent.